Ultrasonic treatment of Dendrobium officinale polysaccharide enhances antioxidant and anti‐inflammatory activity in a mouse D‐galactose‐induced aging model

Abstract Utilization of the biological macromolecule Dendrobium officinale polysaccharide (DOP) as a functional ingredient is limited by its high intrinsic viscosity and molecular weight. The goal of the present study was to improve rheological properties of DOP by ultrasonic treatment. Such a treatment resulted in the degradation of DOP and consequent reduction of rheological properties. Among DOP samples treated with ultrasonication at low (L), medium (M), and high (H) power intensities (25, 50, 75 w/cm2), M‐DOP displayed the highest reactive oxygen species (ROS) and reactive nitrogen species (RNS) radical scavenging activity in vitro. In a mouse D‐galactose (D‐Gal)‐induced aging model, M‐DOP significantly increased activities of antioxidant enzymes and reduced levels of pro‐inflammatory cytokines in liver. Real‐time polymerase chain reaction (RT‐PCR) analysis indicated that M‐DOP upregulated messenger RNA (mRNA) expression of anti‐inflammatory/antioxidant proteins such as Nrf2 (nuclear factor erythroid 2‐related factor), hemeoxygenase‐1 (HO‐1), and NAD(P)H:quinone oxidoreductase (NQO1) in liver. In summary, M‐DOP displayed a strong radical scavenging activity in vitro, and ameliorated liver injury in the mouse aging model through the promotion of Nrf2/HO‐1/NQO1 signaling pathway.

viscosity. Pan et al. (2014) found that viscosity of polysaccharides from four Dendrobium species ranged from 35 to 127 cm 3 /g. Molecular weight (Mw) of the four polysaccharides ranged from 197 to 378 kDa, and high Mw appeared to be associated with high viscosity (He et al., 2018). The high Mw and viscosity of D. officinale polysaccharide (DOP) make it useful as a gelling or thickening agent in food industries. On the other hand, relatively low Mw and viscosity are preferable for bioactive components in functional foods or beverages. Numerous methods have been applied for depolymerization of polysaccharides, including chemical treatment (Mzoughi et al., 2017), enzyme hydrolysis , ultrasonic treatment (Qiu et al., 2019), and γ-irradiation (Wang et al., 2017). Among these, ultrasonic treatment has several advantages: it is rapid, mild, environmentally friendly, and does not involve toxic reagents. In some cases, immunoregulatory (Yao et al., 2015), antioxidant, and/ or antitumor activity (Yan et al., 2016) of polysaccharides is enhanced by ultrasonic treatment. In the present study, rheological properties (i.e., deformation or flow behaviors in response to applied forces) of DOP were improved by ultrasonic treatment.
Reactive oxygen species (ROS), in their normal function as signaling molecules, are involved in the resumption of meiosis following meiotic arrest, and in the activation of apoptotic pathways (Agarwal et al., 2005). However, in unbalanced states in vivo, ROS may be overproduced and lead to oxidative damage, e.g., lipid peroxidation (Fan & Li, 2014), cross-linking and degeneration of biomacromolecules by malondialdehyde (MDA) (Hipkiss et al., 1998), and inflammatory response (Byun et al., 2017). Accumulation of ROS and other free radicals is a major mechanism in aging processes and contributes to brain aging and senile dementia (Salmon et al., 2010). D-galactose (D-Gal), a reducing sugar, is converted to glucose by galactose (Gal)-1-phosphate uridyltransferase and galactokinase at physiological concentration. Excessive D-Gal levels resulted in disordered cellular metabolism, alteration of oxidase activity, and production of oxidative products . The natural aging process was recapitulated in a D-Gal-induced mouse model based on a free radical theory of aging (Çoban et al., 2015). Besides causing oxidative stress, excessive accumulated D-Gal reacts with free amino acid groups of proteins or peptides, with consequent formation of advanced glycation end-products (AGEs) (Zhou et al., 2015).
Numerous studies have demonstrated that ROS and AGEs are key factors contributing to aging processes and related diseases (e.g., liver damage, nephritis, Alzheimer's disease), and other diseases (Palma-Duran et al., 2018;Saleh et al., 2019). D-Gal-induced aging models are therefore used frequently in antiaging pharmacological research.
We applied the ultrasonic treatment to improve the rheological properties of extracted DOP and evaluated structural properties (monosaccharide composition, chemical composition, Mw) during treatment. Radical scavenging activity was examined in vitro to identify the active fraction of DOP following ultrasonic treatment. Biochemical indexes and pro-inflammatory cytokines in serum and liver following DOP treatment were analyzed in a mouse D-Gal-induced aging model to clarify relationships between structure and biological activity of the polysaccharide.

| Materials and reagents
Dendrobium officinale collected at 3 years after greenhouse planting was purchased at a Chinese herbal medicine market and identified by Associate Professor and senior lab master Tong Chen (School of Life Science, Taizhou University). Dextran with various Mw values, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 1,10-phenanthroline, nitrotetrazolium blue chloride, phenazine methosulfate, NADH, sodium nitroprusside, and D-Gal were from Sigma-Aldrich. Other reagents used were of analytical grade.

| D. officinale polysaccharide (DOP) extraction
Dendrobium officinale stems were dried at 105°C for at least 12 h until reaching a constant weight and then ground to powder (50mesh sieve). The powder was extracted in 1000 ml boiling water for 3 h. Extract supernatant was collected by centrifugation (5000 × g, 10 min), and residual powder was subjected to the same extraction process twice. Combined supernatants were added with ethanol (4× volume). Ethanol residue was removed, and precipitates were deproteinized by Sevage method and concentrated by lyophilization to obtain DOP.

| Depolymerization of DOP by ultrasonic treatment
An ultrasound generator (Jiangsu Bode Ultrasound Equipment Co.) was used for ultrasonic treatment. Twenty milliliters of DOP solution (1 mg/ml) was treated with 20 kHz/750 W, and a bottle containing the solution was kept in ice water to avoid overheating. Amplitude parameters 20%, 40%, and 60% were set using the control panel.
Ultrasound power intensities (power per unit area of probe tip) corresponding to these three amplitudes were, respectively, 25, 50, and 75 w/cm 2 , based on the formula described previously (Li et al., 2004). Following ultrasonic treatment, DOP was separated by ultrafiltration membrane (Mw cut-off 30 kDa), concentrated by lyophilization, and subjected to analysis of structural properties and biological activities.

| Intrinsic viscosity analysis
Dendrobium officinale polysaccharide was dissolved to various concentrations (1.0, 1.25, 1.5, 2.0, 2.5 mg/ml), and viscosity of the solutions was measured by Ubbelohde-type viscometer (Hangzhou Zhuoxiang Technology Co.) at 25°C. Each solution was centrifuged (5000 × g, 10 min), filtered using 0.45μm membrane, and elapsed time going through the viscometer was recorded. Intrinsic viscosity was defined as the intercept of ln(ηr/C) and C curves, where ηr = relative viscosity and C = concentration (Yan et al., 2009).

| Chemical composition analysis
Chemical composition analysis was performed to determine contents of carbohydrates (Dubois et al., 1956), proteins (Bradford, 1976, and sulfate radicals (Dodgson & Price, 1962) in DOP, using the methods described in these studies.

| Monosaccharide composition analysis
Monosaccharide composition of DOP was analyzed by gas chromatography (GC), as described in our previous report (Peng et al., 2015).
In brief, 20 mg of polysaccharide was hydrolyzed by H 2 SO 4 and neutralized by BaCO 3 , the obtained samples were reacted with pyridine and acetic anhydride, and the solution of monosaccharide derivatives was analyzed by GC. Inositol was used as internal standard for the calculation of mole percentage.

| Molecular weight analysis
Mw of DOP was measured by high-performance gel filtration chromatography (HPGFC) with Ultrahydrogel Linear Column, and elution by 0.1 M NaNO 3 at a flow rate 0.9 ml/min. DOP solution (20 μl; 0.1%, w/v) was injected, and Mw was recalibrated using dextran with various Mw values.

| Fourier-transform infrared spectroscopy (FTIR) analysis
Dendrobium officinale polysaccharide samples were mixed with potassium bromide (KBr) powder (1:100) and compacted into pellets, which were analyzed by Tensor 27 FT-IR spectrometer (Bruker; Pittcon 2019 Expo). FTIR spectra were recorded in the range of 400-4000 cm −1 , the baseline was adjusted using the computer program supplied with the instrument, and the FTIR profile was plotted using the software program Origin 8.

| Hydroxyl radical scavenging activity
This type of activity was analyzed by the methods described previously . In brief, 2 ml DOP solution (various concentrations) was mixed with 1 ml 1,10-phenanthroline (1.865 mM) and 1 ml FeSO 4 .7H 2 O solution (1.865 mM). The reaction was initiated by adding 1 ml H 2 O 2 (0.03% v/v) and incubated for 1 h at 37°C. Control sample was a reaction mixture without addition of DOP solution, and blank sample was a mixture without addition of H 2 O 2 . Absorbance at 536 nm (A) was measured and applied in the formula:

| Superoxide anion radical scavenging activity
This activity was also determined as described by Wang et al. (2013).
One milliliter of DOP solution (various concentrations) was mixed with 1 ml nitrotetrazolium blue chloride (2.52 mM) and 1 ml NADH (264 mM), added with 1 ml phenazine methosulfate (0.12 mg/ml), and the reaction mixture was incubated for 5 min at 25°C. Control sample was a reaction mixture without addition of DOP. Absorbance at 560 nm was measured and applied in the formula:

| Nitric oxide (NO) radical scavenging activity
This activity was determined as described previously (Yen et al., 2001). As much as 0.25 ml DOP solution (various concentrations) was mixed with 0.25 ml sodium nitroprusside (10 mM, dissolved in phosphate-buffered saline (PBS) solution). The reaction mixture was incubated for 2 h at 25°C and then mixed with an equal volume of the Griess reagent (Sigma) and incubated for 10 min at room temperature. Control sample was a reaction mixture without addition of DOP. Absorbance at 540 nm was measured and applied in the formula:
Mice were maintained at 25°C, 60% relative humidity, automated 12 h light/12 h dark cycle. Following a 1-week period of adaptation, the 40 mice were divided randomly into six groups: control group Nitric oxide radical scavenging activity ( % ) = A control − A sample

| Determination of body weight, biochemical indexes, and cytokine levels
BWs were recorded (simultaneously for all mice) every 2 weeks.

| Real-time quantitative PCR (RT-qPCR)
Livers from the six groups were collected, and total RNA was ex-

| Statistical analysis
Animal experiments were conducted using eight replicates, and other experiments with three replicates. Data were expressed as mean ± SD. Comparisons between two groups were made by oneway analysis of variance (ANOVA) using GraphPad Prism software program, V. 7.0. Differences with p < .05 were considered significant.

| Structural characterization of DOP subjected to three ultrasonic treatments
The high intrinsic viscosity of DOP resulting from high Mw limits its potential application as a functional food ingredient. We depolymerized DOP by ultrasonic treatment (frequency 20 kHz, power 750 w).
DOPs treated with three power intensities (25, 50, 75 w/cm 2 ), respectively, termed L-, M-, and H-DOP, all displayed a sharp reduction of intrinsic viscosity after 20 min treatment, and a fairly constant level thereafter ( Figure S1). Structural differences among L-, M-, and H-DOP were investigated by FTIR, monosaccharide composition, and chemical composition analyses, as described below.
Structural properties were evaluated for L-, M-, and H-DOP treated with or without ultrasonication for 40 min, and for native DOP (N-DOP). Peaks in FTIR spectra were identified as shown in Figure S2, based on a previous study (Huang et al., 2016). The spectra displayed broad characteristic peaks at 3420 cm −1 for hydroxyl stretching vibration, and at 2926 cm −1 for C-H stretching vibration.
Peaks at ~1735 and 1250 cm −1 were attributed to carboxyl and C-O  Glc than for the other five, consistently with previous findings by J.P.
Luo's group (Pan et al., 2014). Ultrasonic treatment of the DOPs had no effect on monosaccharide composition or mole ratio, indicating that it does not alter polysaccharide chains or molecular bonds.
Contents of carbohydrates, proteins, and sulfate radicals for the four DOPs before and after ultrasonic treatment are shown in Table   S2. Protein and sulfate radical contents were highest for N-DOP without ultrasonication. In general, ultrasonic treatment increased carbohydrate content (p < .05) and decreased protein and sulfate radical contents (p < .05). Sulfate radical content was lower for H-DOP than for L-or M-DOP. S. Huang's group observed previously that protein and sulfate radical contents of DOP samples varied depending on the extraction method (He et al., 2018). We observed that ultrasound power intensity is also a key factor affecting DOP chemical composition.

| Antioxidant activity of DOPs
In normal cell metabolism, dynamic redox equilibrium is maintained by the elimination of excess ROS (e.g., hydroxyl radical, superoxide

Mw is an important determinant of antioxidant activity. Previous
studies demonstrated that antioxidant activity varied depending on Mw for various polysaccharides, e.g., in Lycium barbarum (Liu et al., 2021), Codium cylindricum (Yan et al., 2021), and fucoidan (Hou et al., 2012). Reduction of Mw for such polysaccharides enhanced antioxidant activities, including radical scavenging activity and reducing power. Q. Li's group proposed that reduction of Mw resulted in the exposure of more reducing ends for such polysaccharides (Yu et al., 2021). Thus, the high ROS and reactive nitrogen species (RNS) radical scavenging activities we observed for M-DOP may have been due to decreased Mw and increased exposure of reducing ends.

| Liver tissue histopathology
Excessive D-Gal was previously shown to induce liver damage in an aging mouse model (Chen et al., 2020;Li et al., 2019). Similarly, we

| Effect of DOP on the liver function in mouse D-Gal-induced aging model
In vitro experiments revealed strong promotion by M-DOP of ROS and RNS radical scavenging activity (Figure 1). We further inves-

ACK N OWLED G M ENTS
The authors are grateful to Dr. S. Anderson for English editing of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that no competing interests were involved in the research or preparation of the manuscript.